Ribosomes are ubiquitous cellular nanomachines that enable decoding of mRNA and catalyze protein synthesis. Formation of functional ribosomes requires several pre-rRNA processing steps coupled with folding of the pre-rRNA and binding of ribosomal proteins to the rRNA. In humans, dysregulation of these processes leads to diseases related to alterations in normal cell growth and proliferation, including cancer. In recent years, the focus of the field has been to identify the factors involved in ribosome assembly and the specific steps in which they function. However, the exact roles of these proteins have been largely unstudied. These factors include exo- and endonucleases, GTP- and ATPases, RNA helicases, and RNA binding and scaffolding proteins. RNA helicases drive RNA-RNA, RNA-protein, and protein-protein rearrangements and are involved in all processes of RNA metabolism including transcription, splicing, and translation. In the yeast Saccharomyces cerevisiae, over half of the known RNA helicases function in ribosome biogenesis, consistent with this process being highly regulated and timed. The long-term goal of this project is to investigate the functions of the DEAD- box RNA helicase Has1 in 60S ribosome assembly. Using S. cerevisiae, combined genetic, molecular biological, biochemical, and proteomic approaches wil be carried out to address specific questions of Has1 function in two steps of the 60S ribosome assembly pathway. When does Has1 enter and exit preribosomes? Upon which factors does Has1 depend for recruitment into preribosomes? Which ribosomal proteins fail to stably associate with preribosomes when Has1 is depleted? Which assembly factors fail to enter or exit preribosomes when Has1 is depleted? What are the protein targets and cofactors of Has1? What are the RNA targets of Has1? In what local RNA environments does Has1 function? Answers to these questions will be valuable in understanding the exact functions of this protein in ribosome biogenesis and will shed light on more specific roles of RNA helicases in processes of RNA metabolism. Furthermore, because the pathway of ribosome assembly is highly conserved across eukaryotes, the proposed research plan will provide insight into how dysregulation of this process leads to human disease. PUBLIC HEALTH RELEVANCE: Both the Has1 helicase and the pathway of ribosome biogenesis are highly conserved across eukaryotes. Furthermore, the human homolog of Has1 is important for cell growth and its inhibition has been shown to prevent tumor cell proliferation. Therefore, understanding the functions of this helicase in yeast will have important implications in further study of human ribosome biogenesis and disease.!